CA1202918A - Eukaryotic autonomously replicating segment - Google Patents

Abstract

EUKARYOTIC AUTONOMOUSLY REPLICATING SEGMENT

ABSTRACT OF THE DISCLOSURE
Method, compositions and microorganisms involving host eukaryotic chromosomal DNA providing autonomous repli-cating segments (ars) joined to at least one gene capable of expression and replication in a eukaryotic host not naturally joined to the ars. A eukaryotic chromosome is segmented and the segments joined to a gene capable of expression in a yeast and having a phenotypic marker allowing for selection to provide a hybrid DNA molecule. Yeast are transformed with the hybrid DNA molecule and cultured under conditions select-ing for the transformants, whereby the hybrid DNA molecules may be isolated providing a source of the autonomous repli-cating segment. The autonomous replicating segment may then be used in conjunction with one or more genes to transform the host or other eukaryotes to provide for multiple copies of a host gene, an exogenous gene, or for integration or recombination with the host chromosome, as well as expression of the gene(s).
This invention was developed under grants from the National Science Foudation, National Instiute of Health and the United States Public Health Service.

Description

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~ ,, EUKARYOTIC AUTONOMOUSLY REPLICATING SEGMENT

BACKGRO~ND OF T~E INVENTION
5 Field of ~he Invention The ability of extrachromo~omal DNA molecules to replicate autonomously has been utilized to isolate prokaryotic orgins of replication. T~pically, DNA is intro-duced into bacteria via phage infection, conjuga~ion or calcium-mediated transformation. A given DNA molecule will replicate, independent of integration into the host genome, only if it contains an initiation site recognized by the essential replication enz~mes and factors. Propagation of such extra ~hromosomal DNA molecules can be assured by se-lecting for the expression of a linked marker, e.g., a yeneencoding drug resistance ox a gene capable of complementing a host lesion. Thi~ rationale has been used to isolate and define the oxigins of replication of A F and R factor plasmid~, and ~he Salmonella typhimurium and E. coli chromo-somes.
The yeast Saccharomyces cerevisiae is the onlyeukaryote in which a similar selectio~ scheme i~ currently practical. While virions can provide the necessa~y replicat-~_ ing site for replication in eukaryotes, the-intro~ction of - 25 virions,--or portions thereo~,-would~be undesirabl~for many ~pplications employing recombinant ~NA technology. It is ~herefore of substantial importance to find alternative methods for introducing extra chromosomal D~A into a eukaryotic c211, where the extra c~lromosomal DNA will be .. ~

~2~J29~L8 either self-replicating and/or rapidly integrate into the chromosome.
Description of the Prior Art Struhl et al. (1979) Proc. Natl. Acad. ~ i. USA 76, ~ ~ 1035-1039-describes a yeast DNA fragment that is ~ble to transform yeast with high efficiency and behave as a mini-chromosome by replicating-autonomously without integration into the genome. Schexer and Davis (1979) ibid. 76, 4951-4955, describes a method for the stable introduction of foreign sequences into S. cerevisiae chromosomes. The method involves employing vectors which integrate into chromosomal DNA. See also Stinchcomb et al. ~1979) Nature 282, 39-43.
SUMMARY OF THE INVENTION
Eukaryotic autonomous replicating segments are prepared ~y fragmenting host chromosomal DNA and joining the fragments to a marker gene creating a hybrid DNA which allows for the selection of transformants. Yeast or another rapidly growing eukaryote, particularly yeast, is transformed under conditions selecting for expression of the hybrid VNA. The hybrid DNA is isolated ~rom the transformants and the autono-mous xeplicating segments may be further modified and used as a vector joined with genes from the host or other source.
The hybrid will autonomously replicate, allowing for expres-sion of the genes introduced with the autonomous replicating segment~ Subsequently, stable integration of one or more genes in the host chxomosome can be achieved by appropriate construction of the hybrid.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods and compositions are provided for modifying eukaryotic cells to produce cells having multiplicative - copies of an endogenous genej carrying nonreverting mutations ~ resulting in-the loss of a phenotypic property or~expressin~
- - ~ exogenous genes resulting in the production of a~oreign - protein. The genetic change may be--due to the.s~ble..pres-ence of an autonomously replicating hybrid DNA molecule or due to the integration of one or more DNA segments into the host chromosome.

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The hybrid molesules of the subject invention have an "autonomously replicating segment" of DNA derived from a chromosome of a eukaryote (which if from yeast ~.~ less than a lkb pair fragment). The autonomously replica-ting~segment i8 5~ -obtained-by cleaving a chromosome of a eukaryote,~and joining ~ he resulting segments to a DNA segment carrying ~ne or more ~ appropriate-markers, capable of expression in a eukaryotic cell and/or prokaryotic cell. The hybrid DNA is used to transform an appropriate eukaryotic host cell, selecting for transformants due to the phenotypic expression of the marker.
Where a eukaxyotic cell is txansformed, the replication site derived from a eukaryote chromosome will be sufficient for replication of the hybrid DNA in the eukaryote transformant, as well as serving as a marker.
The hybrid DNA may then be isolated, and the eukaryotic DNA segment allowing autonomous replication can be isolated, modified as desired, and then used for transforma-tion of the host or some other similar eukaryotic cell.
I f a DNA sequence homologous to a segment of the host chrsmosome ~nd interrupked by one or more ~enes i~
included in the hybrid DNA cont~;n;ng the autonomous repli-cating segment (ars), the additional genes may be stably integrated into the host chromosome.
The method of obtAi n; ng autonomou~ replicating se~ments (ars~ will be considered first. Eukaryotic DNA is fragmented to provide fragments of a size sufficient to include an entire ars, usually at least about O.lkb, more usually at least about 0.2kb pairs preferably at least about 0.5kb pair~. The segments may be prepared by mechanical disruption of the chromosome or by enzymatic cleavage using ._ one or more restriction enzymes. .~estriction enzymes should `~ . ~.be chosen which do not clea~e ~he ars, although oligomeriza-~ tion would reconstitute -the-ars during preparati~ of..the.
.. . ...hybrid.DNA. Segments ob~ine~.from a eukaryote ~ er than a yeast may provide for replicatio~ in yeast. Thus, the hybrid DNA which is prepared need not have a yeast replicator site and, in fact, such site is desirably absent where the ars gene of inter.~., r. is from an organism other than yeast.

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Where mechanical shearing of the eukaryotic chromo-some is involved, ~he terminii of the DNA se~ments will be modified in accordance to known techniques to allow for ligation to other DNA segments for preparation oft~the hybrid DNA. Modification may include exonuclease degradation, addition of small DNA seguences, either by the ad~ition of ~- individual bases or a preformed se~uence, or the like. Where restriction enzymes are employed for cleaving the chromosome, the same enzyme may produce cohesive ends or blunt ligatable ends which may be used for joining the segments to othex DNA
sequences.
In addition, to the ars gene containing sequence, the hybrid DNA will carry a marker which allows for selection of yeast or other eukaryote transformants. The hybrid may also include a replicon from proXaryotes to allow for multi-plication in a prokaryote host. The markers employed for ~election may be varied widely. Common markers include those imparting antibiotic resistance to such antibiotics as tetracycline, ~mpicillin, penicillin, kanamycin, or the like.
Alternatively, markers used may provide for prototrophy in an auxotroph. Prototrophic capabilities include the expression of enzymes involved in biosynthetic pAthways for the produc-tion of nucleotides, or purine or pyrimidine precursors, such as uracil, adenylic acid, guanylic acid, cytidylic acid, thymidylic acid; or the like, or the production of amino acids, ~uch as lPucine, tryptophan and histidine ox the like;
for the assimilation of re~uired nutrients; or to provide toxin resistance.
Where it is desired to integrate a particular gene into the chromosome of ~he host eukaryote, different tech _ nigues may be employed. In one way, the hybrid DNA includes ~ an altered seguence of a gene present in the host chromosome, - ~ the~alteration being an insertion of one or more .~nesO By - - ~irtue of ~hP homologous se~ences between the al~red gene

3~ and the host chromosome, the inserted genes can be stably introduced into the host chromosome by r~combination. It is further found, that segregants can be obtained where at least a major portion of the DNA other than the altered seguence is IL~ 9~15 lost rom the chromosome. ~nother way is to include in the hybrid DNA a transposable element adjacent to the gene to be integrated~ The transposable element will integr~te into the - host chromosome simultaneously incorporating the ~ene into the host chromos~me. By culturing the transformant cells ~hrough a number of generations, segreyants having the desired gene stably integrated into the host chromosome can be isolated.
Autonomous replication may be achie~ed with a D~A
fragment having a ~ew as lO base pairs, more usually at least about 50 base pairs, more usually at least about lO0 base pairs. The ars gene will be included in a DNA fragment of at least about 0.2kb pair usually at least about 0.5kb pair and usually less than about ~kb pair. For yeast, the ars gene will be a fragment of less than about lkb pair. The ars gene is characterized by being capable of hybridizing with the eukaryote chromosomal DNA from which it was derived and providing high transformation efficiencies and being free of a ma30r portion of the host chromosome. Usually the DNA
fragment containing the axs gene including other ~enes naturally linked to the ars gene will be under about 20kb pairs, u~ually under lOkb pairs and preferably under 5kb pairs.
Depending upon the ~nner of formation of the fragment containing the ars gen~, other genPs may be linked to the ars. Where other genes, particularly structural genes are present, the ars segment including such genes will be greater ~han about lkb pair in size. There will be many situations where the presence of the gene other than ~he ars will be undesirable. The presence of the other gene with its ` "~
- genes controlling expression may be undesirable in many instances, providing expression of an undesired protein, -~undesired sites for-restriction,~unstable transfo~ants, -- - undesired insertion or insertion at an undesired l~cus, and increased probability of the genes of interest being sepa-rated from the ars by recombinational events. It will there-fore be desirable to ~; n; ~; ze the presence of DNA sequences other than the ~rs gene and linkage seguences. The removal of the other genes may be achieved by employing restriction enzymes, ~electing for fragment~ lackin~ the other g~n~s, removing bases by exonuclease digestion, and the like.
: Desirably less than fifty percentj more usually lerss than about twenty percent of the base pairs of the lin~ed gene -~ will be part of the ars containing fragments.
In some instances multimers of ars genes will be present. The number of units may range from about 2 to 4.
The preparation of the ars cont~'n;ng hybrid DNAs will vary widely depending upon the use of the hybrid DNA, the form in which the DNA sequences are available, existing restriction sites as well as restriction sties to be intro-duced and the purpose of the transformation e.g. chromosomal integration.
Conveniently mapped circular DNA from a plasmid, phage, virus o~ chromosome, having at least one restriction site, preferably two or more different restriction sites, can be employed. The circular DNA may have one or more markers, particularly expressing antibiotic resistance or an enzyme essential to a biosynthetic pathway, or such markers may be inserted into a restriction site. Depending on the nature of the restriction site, the restriction site may be modified by removal of bases with exonucleases or addition of bases by DNA polymerases or ligases. The hybrid DNA may be built up ~5 seguentially or by annealing two DN~ segments comprising all the genes of interest. The manner in which the ars contain-ing hybrid DNAs axe generated is conventional and not criti-cal to this invention, although in many instances one scheme will be preferred over another.
The resulting hybrid DNAs are the~ employed for - transformation of an appropriate eukaryote host, desirably, a ~rapidly multiplying eukaryote, ~uch as S. cerevis~ae. Trans-- -formation is performed in a conventional way, of~n employing ; calcium;shock.! Conveniently spheroplasts ar~ emp~yed for the transformation. After transforming the cell~, the resulting culture is then grown in nutrient medium allowing for selection of transformant~. The hybrid DNA may then be isolated from various co:~onjes and tested for hiyh frequency transformation, which is a property of the ars gene. The hybrid DNA may b~ detected by other means, such as hybri-dization and autoradiography, or the like.
once the ars containing hybrid has been ~solated, it may be modified in a number of different ways.~ Where restriction enzymes have been used for preparing the DNA
se~ments resulting in the hybrid DNA, the hybrid DNA may be cleavPd and the segment containing the ars isolated. If desired, the segment may be further restricted, so as to remove DNA unrelated to ~he ars gene or subjected to exonuclease digestion to remove extraneous terminal bases.
The ars gene may then be used to prepare hybrid molecules fox transformation of the host eukaryote, providing enhanced or new genetic capabilities to the host.
By appropriate manipulations, a hybrid can be prepared which will result in the stabl~ introduction of one or more genes into the host eukaryote chromosome. The inte-gration of ~hese genes into the chromosome is achieved by employing a DNA seguence which is homologous with a DNA
seguence of the host chromosome. B~ selection of an appro-priate restriction site in the seguence, the genes to be introduced into the host chromosome can be inserted between the ter~; ni i of the homologous seguence. The altered se-guence i~ then inserted into an appropriate ars containing vector to provide ~he hybrid DNA.
Upon transformation of the host eukaryote with the hybrid DNA, the homologous seguence will be integrated into the host chromosome. Alternatively, due to the presence of the ar~ in the hybrid DNA, the hybrid DNA will be capable of replication in the host eukaryote acti~g as a minichromosome, - ~ and allowing for expression of the genes present on the - hybrid DNA. Where it is not desired to integrate_the genes into-the chromosome, the~hybrid DNA need not have~an homolo-ous sequence with the`host chromosome for integr~tion.
However, integration can occur as a result of mitotic recom-binakion due to the a homology.
The homologous seguence should be at least about a lkb pair section at each end of the altered gene, usually at 9~1~

least about a 1.Skb pair section, preferably at least about a Z.5kb pair section.
Desirably, the homologous sequence may involve genes or sections of a genome with no known funct_on to avoid altered growth characteristics-or additional nutr;tional reguirements for the mutated transformant. Upon ~ntegration of khe altexed gene into the chromosome, except for expres-sion of the new genes~ there would be no change in the pheno-type properties or growth characteristics of the mutated transformant~
Desirably, the homologous seguence should have a high probability of recombination ~ith the host chromosome.
Therefore, certain sequences will prove to be preferred depending on the efficiency of integration. Integration may include additional DNA se~uences other than the altered gene.
This may be a result of the presence of homologous sequences other than the altered gene which serve as a locus for rec~m-bination or the insertion with the altered gene of seguences linked to the altered gene.
To enhance ~h~ ability to select for the cells in which integration has occurred, a marker may be included in the hybrid DNA for concomitant integration with the sene of interest. By selection for the mar~er, the cell population can be reduced to only those cells which have been trans-formed ~nd where the transformants are unstable, only those cells where integration has occurred. The marker will be expressed unstably as unintegrated, autonomously replicating DNA. Selection ~or stablP expression reduces the cell population to only ~hose cells where integr~tion has occurred.
- Segxegants will result from the transformant having - chromosomes from ~hich are excised either portions of the -~ in~egrated DNA other than the altered gene includ~ng ~he - complementary markers or the DNA seguence providi ~ the recombinational locus.
The genes which may be introduced and expressed in the host eukaryote can provide for the production of a wide variety of proteins resulting in desirable phenotypic proper~

, . . . _ ... . . , _ . . . ... ...

1~ 9~3 ties. Expres~ion of the gene~ can provide enzymes for pro-duction of a wide variety of nutrients, such as amino acids, vltam.ins, or the like; for performing a wide vari~.~y of -chemical reactions, such as oxidation, nitrificat~n, reduc-~- tion, hydrolysis, halogenation, or the like; or-p~duction of a wide variety of non-enzymatic proteins, such as hormones, globulins, albumins, collagen, keratin, or the like.
The eukaryotic host can be any of a variety of vertebrates or non-vertebrates, e.g. ~ ~ls, insects, yeast fungi, mold, or the like; or plants, such as trees, deciduous and non-deciduous, vegetables, fruits, and ~ubers.
The following examples are offered by way of illus~
tration and not by way of limitation.
EXPERIMENTAL
I. Preparation of ars genes.
Several pools of hybrid molecules were made by inserting restriction endonuclease-generated segments of dif~erent eukaryotic chromosomal DNAs in YIp5 (YIp5 has a l.lkb fragment contA;n;ng the ura3 gene inserted by dG/dC
homopolymer extensions into the Ava I site of p8R322. Struhl et al. (1979) PNAS USA 76, 1035-1039~. EcoRI was used to fragment N. crassa, D. discoideum, C. elegans, D.
melanogaster and Z. mays DNA. D. melanogaster DNA was also cleaved with ~indIII and E RI simultaneously. With yea~t, BamHI endonuclease was employed to exclude seguence~ from the endogenous yeast plasmid, Scpl, and the yeast ribosomal gene cluster, which are known to transform with high frequency.
After digestion with the appropriate restriction endonuclease(s~, ~he YIp5 and chromosomal DNAs (each at 15-20~g DNA/ml~ were mixed and ligated with O.l~g T4 DNA
-. ligase in 200mM NaCl, 50mN Tris-HCl pH7.4, lOmM MgS04) lmM
- ATP and lOmM dTT at 4C for 1-24~rs. The ligatior~mixture was directly used to transform yeast cells.
~ NN~27 yeast (a ~ 289 ura3-52 ~_2 a~O) was transformed wi~h each separate pool of YIp5-eukaryotic DNA
hybrids~ The procedure of ~; nn~n et al. (1978) PNAS USA 75, 1g29-1933 was employed with ~he following modifications.
Spheroplasts were prepared by treating lODml of an exponen 2~

tially growing culture with 300 units of lyticase for 30min.
at 30C. After treatment with polyethylene glycol, the cells were immediateiy plated in the regeneration agar ~.107-108 - viable spheroplast per plate). Transformation of ~ 27 with 5~ hybrid DNAs c~ntaining both the YIp5 vector and ~ gene -~ results in Ura colonies growing on selective medla. The ~- frequency at which NNY27 was transformed to Ura varied from approximately 50 colonies/~g of YIp5-N. crassa or YIp5-C.
elegans hybrids to 2,000 colonies/~g for the pool of D.
discoideum hybrids (all values represent Ura transformants per mass of YIp5 DNA present in a hybrid pool and are cor-rected for the different transformation efficiencies observed with a different vector YRpl2). Two separate pools of YIp5-D. melanoqaster hybrids constructed by EcoRI cleavage of different DNA preparations yielded 800 and 1000 Ura trans-formants/~g DNA. Moreover, YIp5-D. melanogaster hybrids constructed using HindIII endonuclease generated 600 Ura colonies/~g hybrid DNA upon transformation of NNY27.
Growth rates of yeast transformants in the standard yeast min;r~ medium wère measured using a Klett-Summerson colorimeter. Stability of the transformed phenotype was assessed by diluting saturated cultures 1:1000 into rich media and ~hen det~rm; ni ng the-percentage of cells that r~ined Ura+ by duplicate platings onto nonselective and selective plates. E. coli transformations, rapid DNA prepa-rations, agarose gel electrophoresis, trans~er to nitrocel-lulose paper, and h~bridization with 32P-labeled pBR322 DNA
were carried out with minor modifications of the published procedures. (Struhl et al., supra; Davi~ et al., J. Advanced Bacterial Genetics Laboratory Manual, Cold Spring Harbor - Laboratorie~ Press, Cold Spring Harbor, New York; Southern ? (1975)-J. Mol. Biol.0 98, 503-517 and Rigby et al~ (1977) J.
- - Mol. Bi~l. 113, 237-251). ~
- -- ; -For~rapid!yeast DNA preparations, total~yeast DNA
was prepared from 5ml cultures of cells grown to ~he sta-tionary phase. Yeast cells were harvested and resuspended in O.4ml of 0.9M soxbitol/50mM potassium phosphate, pH7.5, 14mM
2-mercaptoethanol. Lyticase ~25 units) was added and ~/

~2~ 8 spheroplast formation was allowed to proceed for 30min. at 30C. At this sta~e, the procedure for rapid phage DNA
preparations ~Cameron and Davis ~1977) Nucleic Acids Res. 4, 1429-1448) was used with two changes: The ethano~ precipita-5~ -tion was done at room temperature and the re~ulti~g pellet - ~ was resuspenaed in 50-100~1 of lOmM Tris, p~I7.5/l~M EDTA
containing 0.5~y of pancreatic RNase. These preparations yielded approximately l~g of DNA per ml of original culture.
The DNA is of high molecular weight, relatively un-nicked and is cleavable by all restriction enzymes tested.
A~proximately 10 Ura+ transformants were picked randomly from each transformation and their phenotype as-sessed. NNY27 has a generation time of 2.5hrs. in a r; ni m~
medium supplemented with uracil. Doubling times for ~trains that have been transformed to Ura by the YIp5-yeast DNA
hybrids showed a range of generation times of 4 to 8.5hrs.
The N. crassa hybrid pool yielded transformants that gr~w slightly faster with 3 to 4.2hr generation times. The doubling time for transformant6 generated by the o~her eukaryotes varied from 4.5 to 62hrs.
All of the Ura transformants were unstable. After growing approximately 10 generations under nonselective conditions, 95% vr more of each transformed strain lost the Ura character~ There appeared to be a rough correlation between relative instability and growth rate. The tran~for-mants with longer doubling times lost the Ura phenotype more quickly in a rich media.
The state of the DNA responsible ~or the Ura phenotype of the transformant~ was determined. Yeast DNA was 30 purified from Ura+ transformantsO The circular, extra , ~ chromosomal DNA was separated from the linear, high molecular weight, chromosomal DNA by agaro~e gel electropho~esi~D
~ lectrophoresis of the undigested DNA was rarried~out in 0.6%
- - - -agarose, 40mM Tris-0~ 20mM acetic acid~ 2mM ED~A~for 16hrs.
at 1 volt/cm. The yeast chromosomal DNA and endogenou6 plasmid DNA migrated ts separate areas. The gel was trans-ferred to nitr~cellulo~e and then hybridized with approxi-mately 5 ~ 106 cpm 32P-l~beled pBR322 DNA in 50% formamide, , ~

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O.9M NaCl, 50mM sodium phosphate pH7, 5mM EDTA, 0.2% SDS, and 200~g/ml denatured salmon sperm DNA. The washed and dried nitrocellulose filter ~as used to expose Kodak XR-~ X-ray film. Autoradiography was performed for a few da~ at -70C
using a DuPont lightning Plus intensifying screen~
- In each of 65 transformants representing all sources of hybrid DNAs, the transforming hybrid DNA molecules migrate in unigue positions, dis~inct from both the yeast chromosomal DNA and the endogenous yeast plasmid. The mul-tiple bands of hybridizing DNA are most simply explained assupercoiled and nicked circles of monomer, dimer and (in some cases~ trimer forms of the transforming DNA. Such multimers are often produced by the recombination-proficient yeast~
Again, a correlation could be drawn between the intensity of DNA hybridization and the growth rate of each transformant.
The faster the growth rate, the greater the hybridization, indicating that there are more copi.es of the hybrid se~uences where greater hybridi2ation was observed.
II. Int~gration of altered DNA se~uences con-structed ln vitro.
An internal deletion of the h 3 gene ~Struhl eta~. ~1976) PNAS VSA 73, 1471-1475) was constructed ln vitro by removal of a 150-base-pair H dIII fragmen~ i~ YIpl (Struhl et al., i _ 76~ 1035-1039~. YIpl contains the yeast h 3 gene (which complements E. coli hisB mutations) and the ampicillin-resistance gene of pBR322. The plasmid DNA was cleaved with ~indIII and ligated at low concentrationO An ampicillin-resi~tant transformant of E. coli hlsB463 that ;n~r7 Hi~ and had the desired structure was isolated tc~
provide DNA modified against K restriction. The yeast se~
- guences of the resulting plasmid pBR322-Sc2903 confer no -- selectable phenotype on yeast cell6. The yeast u~a3 ~ene was - ~ ligated to the alterPd hls3 seguences-to permit s~ection of - -yeast transformants. YIp5-and p~R322-Sc2903 were-~cleaved with E RI an~ S~lI. The mixture was ligated and used to transform a pyrF E. coli MB1000. By using the complementa-tion of p~rF mutations by the ura3 se~uences of YIp5, cells containing the desired molecules were identified by 2n ampicillin-resi~tant tetracycline-sen~itive PyrF phenotype.

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The resulting plasmid YIp5-Sc2903 was used to transform a uxa3~ strain (SXl-2 ~ ura3 trpl ~10 ~2) selecting Ura . This plasmid integrates by recombination between the Sc2903 DNA sequences in the plasmid a~d the 5~ homologous DNA seguences on chromosome XV~ The v~ctor alone, - ~ without insert DNA,.has not been observed to tran~-form any ~train contain;n~ ura3-52 to Ura+ (less than 1% of the fre guency obtained using a slightly larger his3 fragment in transformation to His ). The integrated structure was demon-s~rated by hybridization to electrophoretically separatedEcoRl-cleaved total yeast DNA according to the method of Southern, supra.
Strain duplications similar to ~he one present in these transformants have been shown to be unstable ~Struhl et al. 9 supra) and segregate cells lacking the vector seguences and one copy of the duplicatio~. In thi6 case, the select-able marker, the ura3 gene is outside the homologous se-guences and segregates with the vector sequences. Therefore, Ura segregants lack vector sequences. Selection for uraf was removed for ten generations by growth on complete medium (~PD~. Nine hundred colonies derived from this culture.were tested for Ura phenotype. Seven such isolates were identi-fied. Each was tested for the presence of vector se~uences and for the DNA ~tructure of the his3 region.
~11 DNA samples were d eaved with B ~ I, coelectro-phoresed in a 1% agarose, Tris-acetate-EDTA gel at 0.6volts~cm for 36hrs~, transferred to nitrocellulosei probed with nick-translated pBR322-Sc26~6 (the his3 BamHI ragment3 and exposed at -70C with Cronex 4 film in a DuPont lightning Plus intensifying screen for a few hours. The hybridization _ spectrum ~howed one of ~he Ura+ transformants with the e~--~ pected~composite of the transforming DNA (8.1kb) and the his3 ~~ deletion (1.6kb~ and the wild-type hls3 ~NA ~1.75kb). A Ura : i~olate 6howed neither the vector seguences nor ~ e wild-type his3 se~uences, but contained the his3 deletion sequence6 present in the transfonming DNA. All seven Ura isolates had lost all vector seguences and three were found to contain a deletion at the his3 locus.

~Z~Z~3~3 . 14 As expected, these three strains were His and did not revert spontaneously at a detectible level (.less than 10 9~ for ~hat marker. Strains containin~ the his3 delekion are stable and can be used in a manner similar to,.any conven-tionally derived hls3 mutation. Because the straIns are also -~ ura3 and no vector DNA sequences are ~Ipresent, thëy can be retransformed for additional modifications.
As a second test for the subject procedure, galactose-inducible seguences were inserted into the site of the his3 deletion on chromosome XV. A hybrid DNA molecule YIp5-Sc2911~ containing a ~.55kh HindIII fragment homologous to galactose-inducible RNA ~St. John and Davis (1979) Cell 16 443-452~ was constructed by insertion into the single ~indIII
ite of YIp5-Sc2903. This DNA was used to transform Sx2-2.
Sx1-2 was crossed with D13-lA (a ~el his3-532 qal2~ to produce a strain o~ somparable transformation efficiency and opposite mating t~pe . This strain is Sx2-2 (a ura3 ~el ~ n isolate wi~h the transforming DNA integrated near his3 was identi~ied. With the subject hybrid DNA, integra~
~0 tion could occur in any one of four sections of yeast DNA
contained in the transforming DNA.
Grow~h for ten generations in non-selective medium was sufficient to yield Ura segregants. ~s expected, bo~h ~is and ~ clones were found. The His i~olates have the ~ se~uences inserted into chromosome 15, replacing a small portion of the hi~3 gene.
Following the proceduxe described above for elec-trophoresis, DNA structure~ of strains used to insert seguences into his3 sites were in~estigated. Of the five ~trains studied, one His segregant did not contain vector 9 - seguences (8.1kb) or wild-t~pe h~s3 seguences ~1.75kb) but did contain the hls3 deletion and qal insertion seguences (4.2kb). - -_ - A-1.4~b pair fragment cont~;n;ng arsl w~ isolated from restriction endonuclease generated ~ragments of S.
cerevisiae yeast chromosomal DNA. The fragments were inserted into Agt-AB by cleaving the A bacteriophage DNA with EcoR`I: endr~nuclease and covalently joining ~he yeast and A DNA
segments wi~h E. coli ~NA ligase.

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The resulting collection of hybrid phage were plated onto grown ~n a lawn o tryptophan auxotrophs (E. coli W3110~C9B30). The bacteria were grown in M9 + 0.2% maltose (M9 - per liter: 6g Na2HP04, 3g KH2 4' g 4 5~- with the following additions lmM MgS04; O.lmM CaCl~; 0.2%
glucose or-maltose~. After sedimenting (8K rpm, 5~in~, the cells were resuspended in lOmM MgS04 to provide about 101 -, cells/ml. The Agt-AB yeast hybrids (2 x 10~) were adsorbed with 2 x 109 E. coli W3110 ~C9830 for 15min at 37~C, and were plated by adding 2.5ml of M9 soft agar (~0.6~) (M9 soft agar - autoclave agar in water and cool to 50~C. For lQ add lOOml lOX M9 salts, O.lml lM CaC12, lml lM MgSO4, lOml ~0%
glucose and for non-selecting amino acid requirements lOml of 4mg/ml each amino acid.) The phage were incubated for 6-40 hours at 37C. Plaques are produced only if the hybrid A phage can complement the bacterial mutation. A hybrids containing yeast DNA fragments were found to complement the trp C1830 mutation. The hybrids are digested with Eco~I
endonuclea~e and electrophoresed on agarose gel (0.7~ See 20 Thomas and Davi~, J. Mol. Biol~ 91, 315 (1974).) A common band of 1.4~b was present in all complementinq phaye. The 1.4kb pair fragment was isol~ted and shown to transform yeast at high efficiency, demonstrating the presence of the arsl gene.
The 1.4kb pair fragment (Sc 4101) was cleaved further as follows. Both PstI and HindIII restriction endo nuclease~ have unique cleavage sites in 5c 4101 and the bacterial vector. Digestion of YR~7 and ~Rp7' SpBR322 with Sc4101 inserted in either orientation) followed by circulari-zation and ligation resulted in deletion of part of the yeast -- seguences, generating Yp411 and Yp413 ~Pstl cleavage) and ? Yp412 and Yp414 (~ dIII cleavage). Fragments containing the - ~ ura3 or-his3~seguences were.-then inserted into ea~h o ~he , our deletions using th~ same restriction endonuc~eases and Yp413/~IS3 and Yp41~fURA3~ were found to be cap ~ le of transforming h 3 and ura3 yeast strains respectively. The arsl seguence is therefore in ~he 850 base pairs between the E RI and ~indIII cleavage sites and deletion of the 200 base pair~ bctween the H dIII and PstI si~e~ i~ an ar~l mutation.

:~o~

In accordance with the subject invention, eukaryotes can be transformed for the introduction of en-hanced genetic capability of an endogenous gene: mutated to inhibit-expression of an endogenous gene by homolq~ous recom-5~ bination with an allele; or provided with novel o~ foreign -~ genetic Gapabilities. One or more of the above changes can be achieved either by providing for an autonomously replicat ing hybrid DNA in the eukaryotic cell or by providing for integration of one or more genes from the h~brid DNA into the eukaryote's chromosome.
The use of the autonomously replicating segment provides many advantages. First, high transformation effi-ciency can be achieved by employing hybrid DNAs containing ars genes. Second, since the ars can be dexived from the host or a different eukaryote compatible with the host, foreign DNA sequences, such as viral seguences to act as vectors, can be avoided.
It was surprising to find that chromosomes from eukaryotes could be segment d and sequences obtai~ed which would be effectlve in allowing autonomous replicatio~ in a eukaryote, dist ~ct from the host chromosomes. Prior to this discovery, it had not been shown that eukaryotes other than yeaæt could provide an ars gene which would permit replica-tion of the ars ge~e and cojoined other DNA segments. How-ever, even with the yeast ~egment, the ars was joined to a structural gene trpl. In addition, it was also discovered that the replication ~ignal6 in other eukaryotes are suffi-ciently ~imilar to the replication 6ignals of S. cerevisiae to permit replication of hybrid ~NAs in yeast.
By employing hybrid DNA cont~n;ng the ars gene, _ greatly enh~nced efficiency of integration of genes can be achieved. Since the hybrid DNA will be ret~;ned ~n the ~ multiplying cells over a number of generations, t~e proba-- - --bili~ty o~ integration is greatly enh~nced. Thus,~ y using the two technigues toge~her - employing the ars gene in addition to an altered gene haviny homologous sequences for integration - one can provide for an improved method ~or integrating DNA segments into a eukaryote chromosome pro-~;~V~918 ducing a stable mutant ~train. Even if integration does notocsur, the hyhrid ~NA can be stably maintained under selec-tive conditions due to the presence of the ars.
Although the foregoing invention has be~n described in some detail by way of illustration and example;:for pur-poses of clarity of understanding, it will be obv~-ous that certain changes and modifications may be practiced within ~he scope of the appended claims.

. - _ ~ i~

Claims (26)

WHAT IS CLAIMED IS:

1. A composition which is a DNA sequence of at least 10 base pairs defining an autonomously replicating segment (ars) of a eukaryote, with the proviso that when said eukaryote is yeast, said autonomously replicating segment is not more than about a lkb pair segment and free of any func-tional structural gene naturally linked to said ars gene.

2. A composition according to Claim 1, wherein said eukaryote is a vertebrate.

3. A composition according to Claim 1, wherein said eukaryote is a non-vertebrate.

4. A composition according to Claim 1, wherein said eukaryote is a plant.

5. A composition according to Claim 1, wherein said eukaryote is other than a yeast.

6. An autonomously replicating segment (ars) comprising a DNA sequence of at least 10 base pairs derived from a eukaryotic host other than yeast prepared by:
(1) fragmenting at least a portion of host eukaryote chromosome containing an ars to provide first DNA
segments;
(2) joining said first DNA segments to second DNA
segments, having a gene capable of expressing a phenotypic property in yeast, to provide hybrid DNAs;
(3) transforming yeast cells with said hybrid DNAs to generate yeast transformants and growing said transformants under selective conditions for said phenotypes to provide a selective transformant culture;

(4) isolating hybrid DNA from said transformant culture, and (5) segmenting said hybrid DNA's to provide DNA
fragments containing said ars.

7. An ars according to Claim 6, wherein said fragmenting is under conditions to generate ars containing DNA fragments substantially free of functional naturally present structural genes.

8. A eukaryotic cell containing hybrid DNA containing an ars as in claim 1, which is endogenous to said eukaryotic cell, with the proviso that when said eukaryotic cell is a yeast cell, said ars is linked to other than structural genes naturally linked to said ars in vivo without intervention of a functional naturally linked structured gene.

9. A eukaryotic cell according to Claim 8, wherein said hybrid DNA contains a gene capable of expression of a phenotypic property in said cell, said gene being derived from a host which does not naturally exchange genetic information with said cell.

10. A method for generating a phenotypic property in a eukaryotic host which comprises:
transforming cells of said eukaryotic host with hybrid DNA comprising a eukaryote ars gene as in claim 1 and a gene capable of expression in said host and expressing said phenotypic property; and growing said cells under conditions where said pheno-typic property is expressed.

11. A method according to Claim 10, wherein said phenotypic property is production of an enzyme.

12. A method according to Claim 11, wherein said enzyme is in the biosynthetic pathway of production of a metabolite.

13. A method according to Claim 11, wherein said phenotypic property is production of a non-enzymatic protein.

14. A method for producing hybrid DNA capable of autonomous replication in a eukaryotic host which comprises:
joining an ars as in claim 1 and having complemen-tary terminii to a DNA segment having complementary terminii.

15. A method according to Claim 14, wherein said complementary terminii are staggered and including the addi-tional step of ligating said complementary terminii.

16. A hybrid DNA comprising a eukaryotic host auto-nomously replicating segment (ars) as in claim 1, and a gene capable of expression in said host and derived from a source which does not normally exchange genetic information with said host, with the proviso that when said host is a yeast, the ars is linked to other than a structural gene naturally linked to said ars gene without intervention of a functional naturally linked structural gene.

17. A hybrid DNA according to Claim 16, wherein said eukaryotic host is a plant.

18. A hybrid DNA according to Claim 17, wherein said plant is corn.

19. A hybrid DNA according to Claim 16, wherein said eukaryotic host is a non-vertebrate.

20. A hybrid DNA according to Claim 16, wherein said eukaryotic host is a vertebrate.

21. A hybrid DNA according to Claims 16, 17 or 18 including a gene expressing a phenotypic property capable of selection.

22. In a method for enhancing the stable inte-gration of a specified gene, capable of expression in cells of a eukaryotic host, into a chromosome of said host, said method including the steps of 1) transforming said cells with a hybrid DNA
comprising said specified gene and a DNA sequence capable of insertion into said chromosome linked to said specified gene; and 2) growing said cells containing said hybrid DNA
under conditions selective for expression of said specified gene stably integrated into said chromosome;
the improvement comprising:
including in said hybrid DNA an ars gene as in claim 1.

23. A method according to claim 22, wherein said insertion sequence is a DNA sequence homologous with a DNA
sequence of said chromosome.

24. A method according to claim 23 wherein said insertion sequence is a transposable element.

25. Eukaryotic host cells resulting from a method according to any of Claims 22, 23 or 24.

26. A eukaryotic cell containing hybrid DNA containing an ars as in Claim 1 which is capable of replication in said eukaryotic cell, with the proviso that when said eukaryotic cell is a yeast cell said ars is linked to other than structural genes naturally linked to said ars in vivo without intervention of a functional naturally linked structural gene.